Microorganisms are present ubiquitously in biological specimens and environmental media suitable for their growth. However, some prove harmful to higher organisms and means for detecting their presence is important to preserve the public health. Many means for detecting various types of microorganisms are available offering various advantages with respect to speed and specificity.
All microorganisms have certain requirements for growth and reproduction. In general, microorganisms require the presence of the following for growth: an energy source such as light or carbon compounds; and a source of raw materials including carbon, nitrogen, sulfur, and phosphorus as well as trace amounts of other minerals. Further, microorganisms must be present in a suitable environment wherein an appropriate temperature, pH, salinity and oxygen concentration is maintained.
A common procedure used to detect the presence of microorganisms involves adding a specimen to a culture medium containing all the necessary elements to support growth. The sample may be natural or pretreated, as by membrane filtration, before being added to the culture medium. The medium may or may not contain chemicals such as antimicrobial agents or antibiotics which suppress the growth of microorganisms other than the target microorganism. Usually, these culture media are sterilized to assure no interference from contaminating microbes, and a rather long incubation period of from twenty-four to forty-eight hours is usually required to grow the microbes to detectable concentrations. Additionally, once growth is detected in these procedures, the target microorganism must be identified using one or more tests specific for a variety of physical or biochemical characteristics unique to the target microbes. These procedures are, therefore, labor intensive and time consuming.
Efforts have been made to simplify and expedite the detection process. Among these efforts have been attempts to measure specific metabolic by-products of individual microorganisms. These methods include: electrical impedance assays, ATP assays, antibody-based assays and carbon-14 labelled substrate assays. Indicators of microbial growth have also been used to monitor growth of target microbes which change color only after growth of the target microbe is detected. These indicators normally react chemically with a metabolic by-product produced by the target microbes resulting in a color change in the medium. Examples of chemicals which change color in the presence of pH changes associated with growth include phenol red, bromocresol blue, and neutral red. For example, Golber, U.S. Pat. No. 3,206,317 uses phenol red, a chemical which changes color in the presence of acidic waste products produced by the target microbe. Berger et al., U.S. Pat. No. 3,496,066 describes the use of compounds which bacteria convert to dyestuffs, e.g., tropinones and dioxans, Bochner, U.S. Pat. No. 4,129,483 describes using a non-biodegradable substance (tetrazolium) which is chemically reduced to produce a color change. In all of these examples, the indicator is a compound which does not serve as a source of a required nutrient.
Edberg, U.S. Pat. No. 4,925,789 describes the use of a nutrient indicator which not only serves as a nutrient source, but also changes color upon being metabolized. The patent, herein incorporated by reference, provides a medium containing a nutrient indicator which, when metabolized by a target bacterium, releases a moiety which imparts a color or other detectable change to the medium. The procedure takes advantage of an enzyme specificity unique to a particular species or groups of bacteria. It suggests using antibiotics to select for growth of the microorganisms targeted and provides specific examples of liquid based assays. Other methods previously used such as Kilian et al., Acta. Path. Microbiol. Scand. Sect.B .sctn.7 271-276 (1979) and Damare et al., J. Food Science 50:1736 (1985) report use of agar-based media without antibiotics.